Acellular nerve grafts supplemented with induced pluripotent stem cell-derived exosomes promote peripheral nerve reconstruction and motor function recovery

Peripheral nerve injury is a great challenge in clinical work due to the restricted repair gap and weak regrowth ability.Herein,we selected induced pluripotent stem cells(iPSCs)derived exosomes to supplement acellular nerve grafts(ANGs)with the aim of restoring long-distance peripheral nerve defects.Human fibroblasts were reprogrammed into iPSCs through non-integrating transduction of Oct3/4,Sox2,Klf4,and c-Myc.The obtained iPSCs had highly active alkaline phosphatase expression and expressed Oct4,SSEA4,Nanog,Sox2,which also differentiated into all three germ layers in vivo and differentiated into mature peripheral neurons and Schwann cells(SCs)in vitro.After isolation and biological characteristics of iPSCs-derived exosomes,we found that numerous PKH26-labeled exosomes were internalized inside SCs through endocytotic pathway and exhibited a proliferative effect on SCs that were involved in the process of axonal regeneration and remyelination.After that,we prepared ANGs via optimized chemical extracted process to bridge 15 mm long-distance peripheral nerve gaps in rats.Owing to the promotion of iPSCs-derived exosomes,satisfactory regenerative outcomes were achieved including gait behavior analysis,electrophysiological assessment,and morphological analysis of regenerated nerves.Especially,motor function was restored with comparable to those achieved with nerve autografts and there were no significant differences in the fiber diameter and area of reinnervated muscle fibers.Taken together,our combined use of iPSCs-derived exosomes with ANGs demonstrates good promise to restore long-distance peripheral nerve defects,and thus represents a cell-free strategy for future clinical applications.

End-to-end and end-to-side neurorrhaphy between thick donor nerves and thin recipient nerves:an axon regeneration study in a rat model

During nerve reconstruction,nerves of different thicknesses are often sutured together using end-to-side neurorrhaphy and end-to-end neurorrhaphy techniques.In this study,the effect of the type of neurorrhaphy on the number and diameter of regenerated axon fibers was studied in a rat facial nerve repair model.An inflow-type end-to-side and end-to-end neurorrhaphy model with nerve stumps of different thicknesses（2：1 diameter ratio） was created in the facial nerve of 14 adult male Sprague-Dawley rats.After 6 and 12 weeks,nerve regeneration was evaluated in the rats using the following outcomes：total number of myelinated axons,average minor axis diameter of the myelinated axons in the central and peripheral sections,and axon regeneration rate.End-to-end neurorrhaphy resulted in a significantly greater number of regenerated myelinated axons and rate of regeneration after 6 weeks than end-to-side neurorrhaphy;however,no such differences were observed at 12 weeks.While the regenerated axons were thicker at 12 weeks than at 6 weeks,no significant differences in axon fiber thickness were detected between end-to-end and end-toside neurorrhaphy.Thus,end-to-end neurorrhaphy resulted in greater numbers of regenerated axons and increased axon regeneration rate during the early postoperative period.As rapid reinnervation is one of the most important factors influencing the restoration of target muscle function,we conclude that end-to-end neurorrhaphy is desirable when suturing thick nerves to thin nerves.

Promoting axonal regeneration following nerve surgery： a perspective on ultrasound treatment for nerve injuries

Nerve injury is often associated with limited axonal regeneration and thus leads to delayed or incomplete axonal reinnervation.As a consequence of slow nerve regeneration,target muscle function is often insufficient and leads to a lifelong burden.Recently,the diagnosis of nerve injuries has been improved and likewise surgical reconstruction has undergone significant developments.However,the problem of slow nerve regeneration has not been solved.In a recent meta-analysis,we have shown that the application of low-intensity ultrasound promotes nerve regeneration experimentally and thereby can improve functional outcomes.Here we want to demonstrate the experimental effect of low intensity ultrasound on nerve regeneration,the current state of investigations and its possible future clinical applications.

Role of adipose tissue grafting and adipose-derived stem cells in peripheral nerve surgery

The application of autologous fat grafting in reconstructive surgery is commonly used to improve functional form.This review aims to provide an overview of the scientific evidence on the biology of adipose tissue,the role of adipose-derived stem cells,and the indications of adipose tissue grafting in peripheral nerve surgery.Adipose tissue is easily accessible through the lower abdomen and inner thighs.Non-vascularized adipose tissue grafting does not support oxidative and ischemic stress,resulting in variable survival of adipocytes within the first 24 hours.Enrichment of adipose tissue with a stromal vascular fraction is purported to increase the number of adipose-derived stem cells and is postulated to augment the long-term stability of adipose tissue grafts.Basic science nerve research suggests an increase in nerve regeneration and nerve revascularization,and a decrease in nerve fibrosis after the addition of adipose-derived stem cells or adipose tissue.In clinical studies,the use of autologous lipofilling is mostly applied to secondary carpal tunnel release revisions with promising results.Since the use of adipose-derived stem cells in peripheral nerve reconstruction is relatively new,more studies are needed to explore safety and long-term effects on peripheral nerve regeneration.The Food and Drug Administration stipulates that adipose-derived stem cell transplantation should be minimally manipulated,enzyme-free,and used in the same surgical procedure,e.g.adipose tissue grafts that contain native adipose-derived stem cells or stromal vascular fraction.Future research may be shifted towards the use of tissue-engineered adipose tissue to create a supportive microenvironment for autologous graft survival.Shelf-ready alternatives could be enhanced with adipose-derived stem cells or growth factors and eliminate the need for adipose tissue harvest.

Emerging issues in peripheral nerve repair

It is today widely acknowledged that nerve repair is now more than a matter of perfect microsurgical reconstruction only and that, to further improve clinical outcome, the involvement of different scientific disciplines is required. This evolving reconstructive/regenerative approach is based on the interdisciplinary and integrated pillars of tissue engineering such as reconstructive microsurgery, transplantation and biomaterials. In this paper, some of the most promising innovations for the tissue engineering of nerves, emerging from basic science investigation, are critically overviewed with special focus on those approaches that appear today to be more suitable for clinical translation.

Experimental nerve transfer model in the neonatal rat

Clinically,peripheral nerve reconstructions in neonates are most frequently applied in brachial plexus birth injuries.Most surgical concepts,however,have investigated nerve reconstructions in adult animal models.The immature neuromuscular system reacts differently to the effects of nerve lesion and surgery and is poorly investigated due to the lack of reliable experimental models.Here,we describe an experimental forelimb model in the neonatal rat,to study these effects on both the peripheral and central nervous systems.Within 24 hours after birth,three groups were prepared:In the nerve transfer group,a lesion of the musculocutaneous nerve was reconstructed by selectively transferring the ulnar nerve.In the negative control group,the musculocutaneous nerve was divided and not reconstructed and in the positive control group,a sham surgery was performed.The animal's ability to adapt to nerve lesions and progressive improvement over time were depict by the Bertelli test,which observes the development of grooming.Twelve weeks postoperatively,animals were fully matured and the nerve transfer successfully reinnervated their target muscles,which was indicated by muscle force,muscle weight,and cross sectional area evaluation.On the contrary,no spontaneous regeneration was found in the negative control group.In the positive control group,reference values were established.Retrograde labeling indicated that the motoneuron pool of the ulnar nerve was reduced following nerve transfer.Due to this post-axotomy motoneuron death,a diminished amount of motoneurons reinnervated the biceps muscle in the nerve transfer group,when compared to the native motoneuron pool of the musculocutaneous nerve.These findings indicate that the immature neuromuscular system behaves profoundly different than similar lesions in adult rats and explains reduced muscle force.Ultimately,pathophysiologic adaptations are inevitable.The maturing neuromuscular system,however,utilizes neonatal capacity of regeneration and seizes a variety of compensation mechanism to restore a functional extremity.The above described neonatal rat model demonstrates a constant anatomy,suitable for nerve transfers and allows all standard neuromuscular analyses.Hence,detailed investigations on the pathophysiological changes and subsequent effects of trauma on the various levels within the neuromuscular system as well as neural reorganization of the neonatal rat may be elucidated.This study was approved by the Ethics Committee of the Medical University of Vienna and the Austrian Ministry for Research and Science(BMWF-66.009/0187-WF/V/3 b/2015)on March 20,2015.

Angiogenesis in tissue-engineered nerves evaluated objectively using MICROFIL perfusion and micro-CT scanning

Angiogenesis is a key process in regenerative medicine generally, as well as in the specific field of nerve regeneration. However, no convenient and objective method for evaluating the angiogenesis of tissue-engineered nerves has been reported. In this study, tissue-engineered nerves were constructed in vitro using Schwann cells differentiated from rat skin-derived precursors as supporting cells and chitosan nerve conduits combined with silk fibroin fibers as scaffolds to bridge 10-mm sciatic nerve defects in rats. Four weeks after surgery, three-dimensional blood vessel reconstructions were made through MICROFIL perfusion and micro-CT scanning, and parameter analysis of the tissue-engineered nerves was performed. New blood vessels grew into the tissue-engineered nerves from three main directions： the proximal end, the distal end, and the middle. The parameter analysis of the three-dimensional blood vessel images yielded several parameters, including the number, diameter, connection, and spatial distribution of blood vessels. The new blood vessels were mainly capillaries and microvessels, with diameters ranging from 9 to 301 μm. The blood vessels with diameters from 27 to 155 μm accounted for 82.84% of the new vessels. The microvessels in the tissue-engineered nerves implanted in vivo were relatively well-identified using the MICROFIL perfusion and micro-CT scanning method, which allows the evaluation and comparison of differences and changes of angiogenesis in tissue-engineered nerves implanted in vivo.

Neuroprotective effects of ginsenoside Rg1-induced neural stem cell transplantation on hypoxic-ischemic encephalopathy

Ginsenoside Rgl is the major pharmacologically active component of ginseng, and is reported to have various therapeutic actions. To determine whether it induces the differentiation of neural stem cells, and whether neural stem cell transplantation after induction has therapeutic effects on hypoxic-ischemic encephalopathy, we cultured neural stem cells in 10-80 ~tM ginsenoside Rgl. Immunohistochemistry revealed that of the concentrations tested, 20 mM ginsenoside Rgl had the greatest differentiation-inducing effect and was the concentration used for subsequent exper- iments. Whole-cell patch clamp showed that neural stem cells induced by 20 jaM ginsenoside Rgl were more mature than non-induced cells. We then established neonatal rat models of hypox- ic-ischemic encephalopathy using the suture method, and ginsenoside Rgl-induced neural stem cells were transplanted via intracerebroventricular injection. These tests confirmed that neural stem cells induced by ginsenoside had fewer pathological lesions and had a significantly better behavioral capacity than model rats that received saline. Transplanted neural stem cells expressed neuron-specific enolase, and were mainly distributed in the hippocampus and cerebral cortex. The present data suggest that ginsenoside Rgl-induced neural stem cells can promote the partial recovery of complicated brain functions in models of hypoxic-ischemic encephalopathy.

Direct muscle neurotization after end-to-end and end-to-side neurorrhaphy An experimental study in the rat forelimb model

The need for the continuous research of new tools for improving motor function recovery after nerve injury is justified by the still often unsatisfactory clinical outcome in these patients. It has been previously shown that the combined use of two reconstructive techniques, namely end-to-side neurorrhaphy and direct muscle neurotization in the rat hindlimb model, can lead to good results in terms of skeletal muscle reinnervation. Here we show that, in the rat forelimb model, the combined use of direct muscle neurotization with either end-to-end or end-to-side neurorrhaphy to reinnervate the denervated flexor digitorum muscles, leads to muscle atrophy prevention over a long postoperative time lapse （10 months）. By contrast, very little motor recovery （in case of end-to-end neurorrhaphy） and almost no motor recovery （in case of end-to-side neurorrhaphy） were observed in the grasping activity controlled by flexor digitorum muscles. It can thus be concluded that, at least in the rat, direct muscle neurotization after both end-to-end and end-to-side neurorrhaphy represents a good strategy for preventing denervation-related muscle atrophy but not for regaining the lost motor function.

Vascularized nerve“grafts”:just a graft or a worthwhile procedure?

The aim of this review is to extrapolate evidence regarding the use of vascularized nerve grafts(VNGs)in peripheral nerve reconstruction and summarize available data on their indications,if any,and clinical applications.A review of the literature via the PubMed database was performed with analysis of ninety-five articles on the experimental and clinical studies of VNGs.Eight relevant questions were selected to be answered about VNGs.VNGs allow faster nerve regeneration and convey a functional advantage under certain clinical conditions such as large nerves,proximal lesions,and nonvascularized recipient beds.Several donor sites are available which have been being divided by body region in this manuscript.VNGs perform better than non-VNGs and provide an advantage in selected cases.However,limited availability and donor site morbidity still limit their application.We foresee a wider application of vascularized nerve allografts to overcome these problems.

Programmable dual responsive system reconstructing nerve interaction with small-diameter tissue-engineered vascular grafts and inhibiting intimal hyperplasia in diabetes

Small-diameter tissue-engineered vascular grafts(sdTEVGs)with hyperglycemia resistance have not been constructed.The intimal hyperplasia caused by hyperglycemia remains problem to hinder the patency of sdTEVGs.Here,inspired by bionic regulation of nerve on vascular,we found the released neural exosomes could inhibit the abnormal phenotype transformation of vascular smooth muscle cells(VSMCs).The transformation was a prime culprit causing the intimal hyperplasia of sdTEVGs.To address this concern,sdTEVGs were modified with an on-demand programmable dual-responsive system of ultrathin hydrogels.An external primary Reactive Oxygen Species(ROS)-responsive Netrin-1 system was initially triggered by local inflammation to induce nerve remolding of the sdTEVGs overcoming the difficulty of nerve regeneration under hyperglycemia.Then,the internal secondary ATP-responsive DENND1A(guanine nucleotide exchange factor)system was turned on by the neurotransmitter ATP from the immigrated nerve fibers to stimulate effective release of neural exosomes.The results showed nerve fibers grow into the sdTEVGs in diabetic rats 30 days after transplantation.At day 90,the abnormal VSMCs phenotype was not detected in the sdTEVGs,which maintained long-time patency without intima hyperplasia.Our study provides new insights to construct vascular grafts resisting hyperglycemia damage.

Isolated tibial nerve injury: a rare presentation

Tibial nerve injury is rare and is always associated with other injuries due to its close association with the other structures.We present a rare case of isolated injury to the tibial nerve where the nerve was avulsed from the middle third of the leg,but all other structures were intact.The nerve was reconstructed with sural nerve grafts.The patient recovered sensation of the sole twelve months following the reconstruction and was able to maintain a normal gait and is living normal life.The results of nerve repairs in lower limbs in general have been poor.The treatment options for such an interesting case are discussed along with the management and outcome of the presented patient.

Computer-aided 3-D reconstruction and measurement of the optic canal and intracanalicular structures

Objective To reconstruct the human optic canal and its inner structures, and to provide detailed knowledge of this region for optic nerve decompression for further understanding on the pathologic mechanisms of indirect optic nerve injury Methods Six optic canals and their inner structures were reconstructed using a computer aided 3 dimensional reconstruction system Quantitative measurement of the canal wall thickness, bony canal transverse area, optic nerve transverse area, dural sheath transverse area, subarachnoid space transverse area, and subarachnoid space volume were done by means of the computer morphometric analysis system The detailed spatial relationship among intracanalicular structures were also carefully identified on the 3 D models Results The thinnest portion of the canal was the middle part of the medial wall (0 45±0 35?mm) and the narrowest space was in the middle part of the optic canal (the transverse area was 18 21±2 50?mm 2) The volume of subarachnoid space which can be considered the compensatory space for distention incurred by the hemorrhage, optic nerve edema, or hematoma was 21 16±4 31?mm 3 At the cranial opening ,the middle part and orbital opening, its transverse area was 4 45±1 12?mm 2, 2 68±1 32?mm 2 and 1 23±0 83?mm 2, respectively Conclusions Since the compensatory space was limited, even a tiny amount of blood or swelling of the nerve may cause optic nerve compression Because the narrowest space was in the middle part of the optic canal and the compensatory space for distention gradually decreases from cranial end to orbital end, the middle part and the anterior part of the optic canal and dural sheath are critical in optic nerve decompression